Knowledge of the fate of released catecholamines from nerve terminals may help to elucidate the role of the sympathetic system in the pathogenesis of hypertension and other diseases in which abnormal sympathetic function has been implicated. For example, experiments carried out with radioactive catecholamines or their precursors have indicated that the deaminated glycol 3,4-dihydroxyphenylglycol (DOPEG), is formed intraneuronally from norepinephrine leaking from granules into the neuronal cytoplasm or from neuronal re-uptake of released norepinephrine, and extraneuronally, together with the deaminated acid 3,4-dihydroxymandelic acid by the action of enzyme monoamine oxidase. Accordingly, it is desirable to be able to quantitatively assay DOPEG to the exclusion of other cathecholamine and catecholamine metabolites.
Various techniques for the measurement of urinary catecholamines and catecholamine metabolites have been developed and applied in many laboratories but most of these methods are non-specific and laborious. The advent of analyses based on radioenzymatic assay techniques make possible the accurate measurement of plasma and tissue catecholamines in small quantities of specimens. See for example the paper by Peuler and Johnson: "Simultaneous Single Isotope Radioenzymatic Assay of Plasma Norepinephrine, Epinephrine and Dopamine," Life Sciences Vol. 21, pp. 625-636 (1977) which describes an assay in which catecholamines are converted to their tritiated O-methylated derivates. Conversion occurs in an incubation mixture by transmethylation with S-adenosyl-L-methionine having a tritiated methyl group, promoted by the transfer enzyme catechol-O-methyl transferase. The tritiated catecholamines are extracted into an organic solvent and separated by thin layer chromatography. Increased specificity is obtained, excluding O-methylated compounds that lack a beta-hydroxyl group by oxidation of at least a portion of the zone scrapings to tritiated vanillin. Measurement of the radioactivity of the tritiated vanillin then constitutes a means for quantitative determination of the chromatographically separated catecholamines.
While the Peuler and Johnson method is useful for measuring catecholamines, the form of assay presented by Peuler and Johnson has not been found useful for the determination of metabolically deaminated products such as 3, 4-dihydroxyphenylglycol. The present disclosure provides a means for assaying 3,4dihydroxyphenylglycol and can be described as a modification of the Peuler and Johnson method. In particular, I have found that by extracting product from the incubation mixture at a pH of 5 or lower, one can decrease the extractability of the catecholamines but increase the extractability of 3-methoxy-4-hydroxyphenylglycol which is the O-methylated derivative of DOPEG. The result is a rapid, specific and inexpensive assaying method for the quantitative measurement of 3, 4-dihydroxyphenylgylcol. The method yields results in less than three hours while one person could easily perform 24 assays in eight hours.
More particularly, a method is provided for assaying 3,4-dihydroxyphenylglycol in a sample containing other catechol compounds, in which an incubation mixture is formed having a pH greater than 7 containing the sample and containing S-adenosyl-L-methionine having a tritiated methyl group and catechol-O-methyl transferase. The mixture is incubated for a time sufficient to transmethylate the 3,4-dihydroxyphenylglycol with the tritiated methyl group to form tritiated 3-methoxy-4-hydroxyphenylglycol. The pH of the incubation mixture is then lowered to 5 or lower and the tritiated 3-methoxy-4-hydroxyphenylglycol is extracted from the lower pH mixture into an organic solvent such as a mixture of toluene and isoamyl alcohol. The tritiated 3-methoxy-4-hydroxyphenylglycol is separated from the organic solvent and isolated, preferably by thin layer chromatography. Also in a preferred step, the separated tritiated 3-methoxy-4-hydroxyphenylglycol is oxidized to tritiated vanillin and combined with a counting solution. By decreasing the pH in the counting solution to about 4.5, extraction is optimal for vanillin which can then be radioactively measured as a quantitative measure of the 3, 4-dihydroxyphenylglycol.
The pH of the incubation mixture can be lowered by the addition of an acid such as glacial acetic acid to achieve for example a pH of 1.5, so that when the incubation mixture is combined with organic solvent having a pH of about 7, the extraction takes place at a pH of 5 or lower. Conducting the extraction at a pH of 5 or lower is critical to success of this assay method as it results in a decrease in extraction of both normetanephrine and metanephrine and a marked increase in the extractability of 3-methoxy-4-hydroxyphenylglycol.